This application is a national stage application of PCT/JP98/03106, filed Jul. 10, 1998, which claims priority to JP 202227, filed Jul. 11, 1997.
This invention relates to a gene expressed specifically in differentiated chondrocytes originating in human (or human chondrocytes), a protein encoded by the gene, an antibody capable of binding to the protein, a method for culturing human chondrocytes in a differentiated state, and human chondrocytes that have been cultured by the method.
Searching for genes expressed specifically in chondrocytes in a differentiated state and analysis of the properties of the chondrocytes are not only important in analyzing the mechanism of differentiation and degeneration of cartilage, but also are indispensable for developing gene therapy for osteoarthritis and rheumatoid arthritis.
However, any method for monolayer culturing human chondrocytes in a differentiated state has not yet been established, although culture systems for rabbit or chicken chondrocytes in a differentiated state have been developed. (Kato et al. Proc. Natl. Acad. Sci. USA 85, 9552-9556 (1988); Oakes et al. J. Embryol. Exp. Morphol. 38, 239-263 (1977).) It is recognized that human chondrocytes maintain their differentiated phenotype in agarose gel (Benya P.D. and Shaffer J.D., Cell 30, 215-224 (1982)), but that they easily lose the differentiated phenotype in the monolayer culture which facilitates handling of cells. Accordingly, it is difficult to search for genes that are expressed specifically in human chondrocytes in a differentiated state thereof; there has not been provided any cell culture system useful in analyzing the properties of the chondrocytes in a differentiated state thereof.
A principle object of this invention is to establish a method of monolayer culture for human chondrocytes in a differentiated state and further to obtain a gene expressed specifically in the chondrocytes in a differentiated state thereof.
The present inventors found that chondrocytes could be cultured in a differentiated state by culturing the chondrocytes in the presence of a certain compound; and in addition, they searched for genes having a distinction in expression between differentiated chondrocytes and dedifferentiated chondrocytes and discovered those which were specifically expressed in the former. Thus, this invention has been accomplished.
Particularly, this invention provides a DNA encoding a protein defined in (a) or (b) as described below: the DNA may be referred to as xe2x80x9cDNA of this (the) inventionxe2x80x9d hereinbelow.
(a) A protein comprising an amino acid sequence set forth in SEQ ID NO: 2.
(b) A protein comprising an amino acid sequence derived from the amino acid sequence set forth in SEQ ID NO: 2 by deletion or substitution of one or more amino acids therefrom, or by addition of one or more amino acids thereto and capable of binding to nucleotide sequence CANNTG and/or nucleotide sequence CACNAG upon formation of a dimer,
wherein the amino acid sequence of a part of said protein corresponding to an amino acid sequence of from amino acid no. 51 to amino acid no. 108 in SEQ ID NO: 2 is provided with not less than 85% of homology to the amino acid sequence of from amino acid no. 51 to amino acid no. 108 in SEQ ID NO: 2. Preferably, DNA of this invention is a DNA defined in the following (c) or (d):
(c) A DNA comprising a nucleotide sequence of from nucleotide no. 207 to nucleotide no. 1442 of the nucleotide sequence set forth in SEQ ID NO: 1 or a complementary nucleotide sequence thereto.
(d) A DNA capable of hybridizing to the DNA defined in (c) under stringent conditions.
This invention also provides a protein encoded by DNA of the invention, as well as an antibody capable of binding to the protein.
Further, the invention provides a method for culturing human chondrocytes, which comprises monolayer culturing the chondrocytes in the presence of a membrane-permeable cAMP analog in an amount sufficient to cause the chondrocytes to maintain a differentiated state thereof as cartilage: the method may be referred to as xe2x80x9cthe culturing method of this (the) inventionxe2x80x9d hereinbelow. Preferably, the membrane-permeable cAMP analog is dibutyryl cAMP (which may be denoted xe2x80x9cdbcAMPxe2x80x9d hereinbelow).
Still further, the invention provides human chondrocytes that have been cultured by the culturing method of the invention and that possess the properties defined in the following (1)-(3):
(1) Exhibit a spherical shape and are abundant in extracellular matrix;
(2) Can be stained with toluidine blue satisfactorily ; and
(3) DNA of the invention is expressed therein.
The DNA of this invention is believed to encode a novel transcription factor of the basic helix-loop-helix type (bHLH), and is predicted to play an important role such as the regulation of expression of various genes in the differentiation of cartilage. Therefore, a DNA of this invention, a protein encoded by the DNA, and an antibody capable of binding to the protein are useful in the analysis of mechanism of the differentiation and degeneration of cartilage, a well as in the development of gene therapy for osteoarthritis and rheumatoid arthritis.
According to the culturing method of this invention, chondrocytes can be monolayer cultured in a good differentiated state, which makes it easy to search for genes having a distinction in expression between chondrocytes in a differentiated state thereof and chondrocytes in a dedifferentiated state thereof: namely, to search for genes expressed specifically in chondrocytes in a differentiated state thereof. Also, an analysis of the properties of chondrocytes in a differentiated state thereof will be facilitated.